An immunoglobulin degrading enzyme

Abstract

The present disclosure relates to the field of biotechnology, and in particular to an immunoglobulin degrading enzyme. The nucleic acid molecule encoding the immunoglobulin degrading enzyme, the recombinant plasmid expressing the immunoglobulin degrading enzyme, or the host cell expressing the immunoglobulin degrading enzyme provided by the present disclosure can realize the biosynthesis of the immunoglobulin degrading enzyme, and during the biosynthesis process, the immunoglobulin degrading enzyme will not cause the autolysis of the host cell, so that the host cell can maintain a high concentration for a long time in the synthesis system, thereby improving the production scale of the immunoglobulin degrading enzyme.

Description

Technical Field

[0001] The present disclosure relates to the field of biotechnology, and in particular to an immunoglobulin degrading enzyme. Background Art

[0002] Immunoglobulin G (IgG) is the main antibody component of serum, accounting for about 75% of serum immunoglobulins. It plays a major protective role in the body's immunity and can effectively prevent infectious diseases. In addition to its protective role, IgG is also related to diseases. In some autoimmune diseases, IgG antibodies react with the body's own molecules, and in organ transplantation, IgG can cause acute transplant rejection.

[0003] IdeS (Immunoglobulin G-degrading enzyme of Streptococcus pyogenes S.pyogenes ) is an extracellular cysteine ​​protease produced by the human pathogen Streptococcus pyogenes (GAS). IdeS catalyzes a single proteolytic cleavage in the lower hinge region of all subclasses of human IgG heavy chains. IdeS effectively cleaves IgG into Fc and F(ab′)2 fragments through a two-stage mechanism. In the first stage, one (first) IgG heavy chain is cleaved to produce a single cleaved IgG (scIgG) molecule with a non-covalently bound Fc molecule. The scIgG molecule is actually an intermediate product that retains the remaining (second) heavy chain of the original IgG molecule. In the second stage of the mechanism, the second heavy chain is cleaved by IdeS to release the F(ab′)2 fragment and the homodimeric Fc fragment, helping GAS to escape antibody-mediated phagocytosis and cellular action, thereby weakening the host immune system's killing of GAS. IdeE is derived from Streptococcus equi subsp. zooepidemicus ( Streptococcus equi ssp.equi ), which is a pathogen of horses (Jonas Lannergård, Bengt Guss. FEMS Microbiol Lett, 2006, 262: 230-235). IdeE and IdeS cleave IgG at exactly the same position, with high reproducibility and specificity, and have very similar substrate ranges.

[0004] In view of the application of the above-mentioned immunoglobulin lysing enzymes, there is a great demand for immunoglobulin degrading enzymes and their large-scale production. Summary of the invention

[0005] The present disclosure provides a novel immunoglobulin degrading enzyme. In the process of using host cells to express the degrading enzyme, the degrading enzyme will not cause autolysis of the host cells, thereby enabling large-scale production of the immunoglobulin degrading enzyme.

[0006] A first aspect of the present disclosure provides an immunoglobulin degrading enzyme, wherein the immunoglobulin degrading enzyme comprises an amino acid sequence selected from the group consisting of:

[0007] (a) the amino acid sequence shown in SEQ ID NO: 2;

[0008] (b) an amino acid sequence having one or more amino acid substitutions, additions and / or deletions compared to SEQ ID NO: 2;

[0009] The immunoglobulin degrading enzyme does not cause autolysis of the host cells when expressed in the host cells.

[0010] In an alternative embodiment, during the expression of the immunoglobulin degrading enzyme, the OD of the host cell is 600 greater than 40, 50, 60, 70, 80, 90, 100, 110 or 120, and during the expression process, the OD of the host cell 600 The value will not decrease.

[0011] In an alternative embodiment, the immunoglobulin degrading enzyme is expressed intracellularly in a host cell.

[0012] The second aspect of the present disclosure provides a nucleic acid molecule encoding the immunoglobulin degrading enzyme of the first aspect, wherein the nucleic acid molecule contains a nucleotide sequence selected from the group consisting of:

[0013] (a) the nucleotide sequence shown in SEQ ID NO: 3; or,

[0014] (b) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence identity with the nucleotide sequence shown in SEQ ID NO: 3;

[0015] (c) a nucleotide sequence having one or more nucleotide substitutions, additions and / or deletions compared to SEQ ID NO: 3.

[0016] In a third aspect, the present disclosure provides an expression vector comprising the nucleotide sequence of the nucleic acid molecule described in the second aspect.

[0017] In a fourth aspect of the present disclosure, a host cell is provided, which comprises the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect, and the host cell is a bacterial cell or a fungal cell.

[0018] In an alternative embodiment, the bacterial cell comprises an E. coli cell and the fungal cell comprises a yeast cell.

[0019] In a fifth aspect, the present disclosure provides a method for intracellularly expressing the immunoglobulin degrading enzyme described in the first aspect, comprising (a) or (b):

[0020] (a) transforming the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect into a host cell, culturing the host cell, and then isolating and obtaining the immunoglobulin degrading enzyme;

[0021] (b) Cultivating the host cell described in the fourth aspect, and then isolating and obtaining the immunoglobulin degrading enzyme.

[0022] In an optional embodiment, the method comprises the following steps: (a) selecting a single clone of host cells; (b) culturing the monoclonal host cells in seed liquid; (c) transferring the monoclonal host cells of seed culture to a fermenter for culture; (d) inducing the monoclonal host cells to express an immunoglobulin degrading enzyme; and optionally (e) collecting the expressed immunoglobulin degrading enzyme.

[0023] The sixth aspect of the present disclosure provides a composition comprising the immunoglobulin degrading enzyme of the first aspect, wherein the composition further comprises an optional pharmaceutically acceptable carrier or excipient.

[0024] In an optional embodiment, the composition further comprises: an antibody or a protein containing an Fc fragment;

[0025] Wherein, the target of the antibody is selected from the following group: cell surface protein, cytokine, hormone, enzyme, intracellular messenger, intercellular messenger or immune checkpoint.

[0026] In an optional embodiment, the composition further comprises: a viral vector drug, and a drug that reduces blood IgG levels;

[0027] Wherein, the viral vector drug is selected from at least one of the following groups: oncolytic virus, gene therapy virus and viral vector vaccine;

[0028] Wherein, the drug for reducing blood IgG level is selected from at least one of the following groups: FcRn antibody, and Fc fragment variant with high affinity to FcRn.

[0029] A seventh aspect of the present disclosure provides a kit, the kit comprising:

[0030] (1) the immunoglobulin degrading enzyme described in the first aspect; and

[0031] (2) one or more selected from the group consisting of: (a) a pharmaceutically acceptable carrier or excipient; (b) an antibody or a protein containing Fc; and / or,

[0032] (3) a viral vector drug selected from an oncolytic virus, a gene therapy virus or a viral vector vaccine; and / or,

[0033] (4) A drug for lowering blood IgG levels, wherein the drug for lowering blood IgG levels is selected from FcRn antibodies, or Fc fragment variants with high affinity to FcRn.

[0034] In an eighth aspect, the present disclosure provides another kit, comprising a medicine box A and a medicine box B;

[0035] Wherein, the drug kit A contains the immunoglobulin degrading enzyme described in the first aspect,

[0036] Wherein, the medicine box B contains one or more selected from the following groups:

[0037] (1) a pharmaceutically acceptable carrier or excipient; (2) an antibody or a protein containing Fc; and / or (3) a viral vector drug; and / or (4) a drug that reduces blood IgG levels;

[0038] Wherein, the viral vector drug is selected from oncolytic virus, gene therapy virus or viral vector vaccine; the drug for reducing blood IgG level is selected from FcRn antibody, or Fc fragment variant with high affinity to FcRn.

[0039] In a ninth aspect, the present disclosure provides a method for reducing the blood IgG level of a subject, comprising administering to the subject an effective dose of the immunoglobulin degrading enzyme described in the first aspect or the composition described in the sixth aspect.

[0040] By using the nucleic acid molecules encoding the immunoglobulin degrading enzyme provided by the present invention, the recombinant plasmid expressing the immunoglobulin degrading enzyme, or the host cell expressing the immunoglobulin degrading enzyme, the biosynthesis of the immunoglobulin degrading enzyme can be achieved. In addition, during the biosynthesis process, the immunoglobulin degrading enzyme will not cause autolysis of the host cells, so that the host cells can maintain a high concentration for a long time in the synthesis system, thereby improving the production scale of the immunoglobulin degrading enzyme. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The electrophoresis results of the expression products of the shake flask fermentation supernatant of the IdeE-1 expression strain in Example 1;

[0042] Figure 2 The electrophoresis result of the expression product of IdeE-2 expression fermentation cell lysis broth in Example 2;

[0043] Figure 3a OD of IdeE-1 expression strain in 5L fermenter at different induction times 600 Condition;

[0044] Figure 3b OD of IdeE-2 expression strain in 5L fermenter at different induction times 600 Condition;

[0045] Figure 3c OD of IdeE-1 expression strain in 5L fermenter at different induction times 600 and changes in turbidity of fermentation supernatant;

[0046] Figure 4 The electrophoresis comparison results of the expression products of IdeE-1 and IdeE-2 expression strains in 5L fermenter at different induction times;

[0047] Figure 5 Changes in the level of IgG degradation in vivo at a dose of 0.25 mg / kg IdeE-2 (KJ103). DETAILED DESCRIPTION

[0048] I. Functional polypeptides with immunoglobulin degrading enzyme activity

[0049] In one or some specific embodiments, the present disclosure provides an immunoglobulin degrading enzyme, wherein the immunoglobulin degrading enzyme comprises an amino acid sequence selected from the group consisting of:

[0050] (a) the amino acid sequence shown in SEQ ID NO: 2;

[0051] (b) an amino acid sequence having one or more amino acid substitutions, additions and / or deletions compared to SEQ ID NO: 2;

[0052] The immunoglobulin degrading enzyme is a mutant of IdeE, and when expressed in a host cell, the immunoglobulin degrading enzyme does not cause autolysis of the host cell.

[0053] In one or certain embodiments, the amino acid sequence of the immunoglobulin degrading enzyme is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in SEQ ID NO: 2.

[0054] In an optional embodiment, during the expression of the immunoglobulin degrading enzyme, the OD600 of the host cells is greater than 40, 50, 60, 70, 80, 90, 100, 110 or 120, and during the expression process, the OD600 value of the host cells does not decrease.

[0055] In some embodiments, the OD of the cells / cells during the expression of the immunoglobulin degrading enzyme of the present disclosure is 600 No decrease will occur after being higher than 40, 50, 60, 70, 80 or 100; for example, during the expression of the immunoglobulin, the OD of the bacteria / cells 600 After it is higher than 70, it will not decrease; for example, during the expression of the immunoglobulin, the OD of the bacteria / cells 600 After it is higher than 80, it will not decrease; for example, during the expression of the immunoglobulin, the OD of the bacteria / cells 600 Once it is above 100, no decrease will occur.

[0056] In some embodiments, during the expression of the immunoglobulin degrading enzyme of the present invention, for example, during 4 hours, 8 hours, 12 hours, 16 hours, 24 hours or longer of induced expression, the OD value of the bacterial cell is 600 No decline will occur above 40, 50, 60, 70, 80 or 100.

[0057] In an alternative embodiment, the immunoglobulin degrading enzyme is expressed intracellularly in a host cell.

[0058] In one or some embodiments, the polypeptide disclosed herein is preferably produced by genetic engineering recombination.

[0059] The second aspect of the present disclosure provides a nucleic acid molecule encoding the immunoglobulin degrading enzyme of the first aspect, wherein the nucleic acid molecule contains a nucleotide sequence selected from the group consisting of:

[0060] (a) the nucleotide sequence shown in SEQ ID NO: 3; or,

[0061] (b) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% but less than 100% sequence identity with the nucleotide sequence shown in SEQ ID NO: 3;

[0062] (c) a nucleotide sequence having one or more nucleotide substitutions, additions and / or deletions compared to SEQ ID NO: 3.

[0063] In a third aspect, the present disclosure provides an expression vector comprising the nucleotide sequence of the nucleic acid molecule described in the second aspect.

[0064] In a fourth aspect of the present disclosure, a host cell is provided, which comprises the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect, and the host cell is a bacterial cell or a fungal cell.

[0065] In an alternative embodiment, the bacterial cell comprises an E. coli cell and the fungal cell comprises a yeast cell.

[0066] In a fifth aspect, the present disclosure provides a method for intracellularly expressing the immunoglobulin degrading enzyme described in the first aspect, comprising (a) or (b):

[0067] (a) transforming the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect into a host cell, culturing the host cell, and then isolating and obtaining the immunoglobulin degrading enzyme;

[0068] (b) Cultivating the host cell described in the fourth aspect, and then isolating and obtaining the immunoglobulin degrading enzyme.

[0069] Some aspects of the present disclosure provide a method for intracellularly expressing the immunoglobulin degrading enzyme of the present disclosure, comprising the following steps:

[0070] (a) selecting a single clone of the host strain of the present disclosure;

[0071] (b) carrying out seed culture of monoclonal host cells;

[0072] (c) transferring the monoclonal host cells from the seed culture to a fermenter for culture;

[0073] (d) inducing the monoclonal host cell to express an immunoglobulin degrading enzyme; and

[0074] (e) optionally collecting the expressed immunoglobulin degrading enzyme.

[0075] The sixth aspect of the present disclosure provides a composition comprising the immunoglobulin degrading enzyme of the first aspect, wherein the composition further comprises an optional pharmaceutically acceptable carrier or excipient.

[0076] In an optional embodiment, the composition further comprises: an antibody or a protein containing an Fc fragment;

[0077] Wherein, the target of the antibody is selected from the following group: cell surface protein, cytokine, hormone, enzyme, intracellular messenger, intercellular messenger or immune checkpoint.

[0078] In an optional embodiment, the composition further comprises: a viral vector drug, and a drug that reduces blood IgG levels;

[0079] Wherein, the viral vector drug is selected from at least one of the following groups: oncolytic virus, gene therapy virus and viral vector vaccine;

[0080] Wherein, the drug for reducing blood IgG level is selected from at least one of the following groups: FcRn antibody, and Fc fragment variant with high affinity to FcRn.

[0081] A seventh aspect of the present disclosure provides a kit, the kit comprising:

[0082] (1) the immunoglobulin degrading enzyme described in the first aspect; and

[0083] (2) one or more selected from the group consisting of: (a) a pharmaceutically acceptable carrier or excipient; (b) an antibody or a protein containing Fc; and / or,

[0084] (3) a viral vector drug selected from an oncolytic virus, a gene therapy virus or a viral vector vaccine; and / or,

[0085] (4) A drug for lowering blood IgG levels, wherein the drug for lowering blood IgG levels is selected from FcRn antibodies, or Fc fragment variants with high affinity to FcRn.

[0086] In an eighth aspect, the present disclosure provides another kit, comprising a medicine box A and a medicine box B;

[0087] Wherein, the drug kit A contains the immunoglobulin degrading enzyme described in the first aspect,

[0088] Wherein, the medicine box B contains one or more selected from the following groups:

[0089] (1) a pharmaceutically acceptable carrier or excipient; (2) an antibody or a protein containing Fc; and / or (3) a viral vector drug; and / or (4) a drug that reduces blood IgG levels;

[0090] Wherein, the viral vector drug is selected from oncolytic virus, gene therapy virus or viral vector vaccine; the drug for reducing blood IgG level is selected from FcRn antibody, or Fc fragment variant with high affinity to FcRn.

[0091] In a ninth aspect, the present disclosure provides a method for reducing the blood IgG level of a subject, comprising administering to the subject an effective dose of the immunoglobulin degrading enzyme described in the first aspect or the composition described in the sixth aspect.

[0092] II. Preparation of functional polypeptides with immunoglobulin degrading enzyme activity

[0093] The present disclosure provides a nucleotide encoding the immunoglobulin degrading enzyme as described above.

[0094] Furthermore, the present disclosure also provides a vector comprising a nucleic acid molecule encoding the immunoglobulin degrading enzyme. The vector may also comprise an expression control sequence operably linked to the sequence of the nucleic acid molecule to facilitate the expression of the protein or mutant.

[0095] A variety of suitable nucleic acid molecules encoding immunoglobulin degrading enzymes as described above are suitable for use in the present disclosure. The sequences mentioned in the examples below are suitable for use in the methods of the present disclosure. It should be understood that when the amino acid sequence of a protein or polypeptide is provided, those skilled in the art can easily determine the nucleic acid molecule encoding it.

[0096] A variety of suitable vectors can be used, such as some vectors used for cloning and expression of mammals, bacteria, fungi, and yeast, such as pET, Pouwels, etc., described in Cloning Vectors: A Laboratory Manual (Elsevier latest edition). In a preferred embodiment of the present disclosure, the vector is a vector suitable for Escherichia coli cells.

[0097] In some embodiments, the vector may be some viral vectors, such as but not limited to retroviral vectors, bacteriophage vectors, adenoviral vectors, herpes simplex virus (HSV) vectors, AAV vectors, or lentiviral vectors.

[0098] Expression vectors include proteins or mutant DNA sequences that are connected with suitable transcription and translation regulatory sequences, such as mammals, microorganisms, viruses or insect genes. Regulatory sequences include transcription promoters, operators, enhancers, ribosome bind sites or control transcription and translation initiation and termination of suitable sequences. When polypeptides as above or mutants need regulatory sequence functions, suitable regulatory sequences are connected. Like this, promoter sequences are connected to the DNA sequence front end of the coded protein or mutant. The ability to replicate in the host cell is usually controlled by the replication origin. The screening gene that is used for transformant identification also can be added to the expression vector.

[0099] In addition, the signal peptide sequence can be fused to the polypeptide or mutant coding sequence so that the translated protein or mutant can be secreted outside the cell. The signal peptide can enhance the host cell to secrete the chimeric polypeptide outside the cell. The signal peptide can be cut off during the process of secretion of the polypeptide from the cell.

[0100] The present disclosure also provides an expression system (e.g., a host cell) for expressing the immunoglobulin degrading enzyme as described above, wherein the expression system contains the expression vector described in the present disclosure or the polynucleotide described in the present disclosure integrated with an exogenous gene in the genome. Any cell suitable for expression by the expression vector can be used as a host cell, for example, the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell, including but not limited to Escherichia coli; Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast, filamentous fungi, plant cells; insect cells of Drosophila S2 or Sf9; animal cells such as CHO, COS, HEK293 cells, or Bowes melanoma cells, etc. A combination of one or more of the above. The method for constructing the expression system should be known to those skilled in the art, for example, it can be a combination of one or more of the above including but not limited to chemical transformation, microinjection, gene gun, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation, etc. As a preferred embodiment of the present disclosure, the expression system is a prokaryotic expression system, such as an Escherichia coli expression system, a Bacillus subtilis expression system, etc. In a more specific embodiment, the Escherichia coli cell is BL21, BL21 (DE3), BL21 (DE3) pLysS, BL21 (DE3) pLysE, BL21 Star (DE3), BL21 Star (DE3) pLysS, Lemo21 (DE3), T7 ExpresslysY, T7 Express lysY / Iq, SHuffle, Origami, Rosetta, HMS174, etc.

[0101] Methods for producing immunoglobulin degrading enzymes as described above are also included in the present disclosure. The methods include culturing recombinant cells containing nucleic acids encoding immunoglobulin degrading enzymes as described above. The methods may include allowing the cells to express the encoded immunoglobulin degrading enzymes as described above, and renaturing the expressed immunoglobulin degrading enzymes as described above. The products of the methods are also protected.

[0102] In one or some embodiments, the step of intracellular or extracellular expression of the immunoglobulin degrading enzyme includes: selecting bacterial cells or fungal cells as host cells, transforming the nucleic acid molecules or expression vectors described in the present disclosure into the host cells, constructing a monoclonal strain expressing the immunoglobulin degrading enzyme, selecting the monoclonal strain for seed liquid culture, fermentation tank expansion culture and induced expression in sequence, and collecting the immunoglobulin degrading enzyme after fermentation is completed.

[0103] In one or some embodiments, the step of intracellular expression of the immunoglobulin degrading enzyme includes: selecting bacterial cells or fungal cells as host cells, transforming the nucleic acid molecules or expression vectors described in the present disclosure into host cells, constructing a monoclonal strain expressing the immunoglobulin degrading enzyme, inoculating the monoclonal strain on an agarose plate, picking a single colony, inoculating it into a test tube / shake flask containing a culture medium, continuing to culture with or without transferring it to a larger volume of shake flask / fermenter, inoculating the cultured bacterial liquid into a fermenter of a certain volume for culture, adding an inducer or other induction methods for induction, continuing to culture until the end of fermentation, and collecting samples.

[0104] In one or some embodiments, the step of intracellular expression of the immunoglobulin degrading enzyme is: selecting bacterial cells or fungal cells as host cells, transforming the nucleic acid molecules or expression vectors described in the present disclosure into host cells, constructing a monoclonal strain expressing the immunoglobulin degrading enzyme, inoculating the monoclonal strain on an LB agarose plate containing 100 μg / ml ampicillin, and culturing at 37°C overnight until colonies grow. Pick a single colony, inoculate it in 3 ml of LB medium containing 100 μg / ml ampicillin, and culture it at 37°C and 250 rpm overnight. Take 500 μl of the overnight cultured bacterial solution and inoculate it in 50 ml of LB medium containing 100 μg / ml ampicillin. After culturing at 37°C for 2 to 4 hours, add 0.1 mM isopropylthiogalactoside (IPTG) for induction, and continue to induce and culture overnight.

[0105] In one or some embodiments, the step of intracellular expression of the immunoglobulin degrading enzyme is: selecting bacterial cells or fungal cells as host cells, transforming the nucleic acid molecules or expression vectors described in the present disclosure into host cells, constructing a monoclonal strain expressing the immunoglobulin degrading enzyme, inoculating the monoclonal strain on an LB agarose plate containing 100 μg / ml ampicillin, and culturing at 37°C overnight until the colonies grow. Pick a single colony, inoculate it in 3ml LB medium containing 100 μg / ml ampicillin, and culture it overnight at 37°C and 250 rpm. Take 500 μl of the overnight cultured bacterial solution and inoculate it in 50ml LB medium containing 100 μg / ml ampicillin. After culturing overnight, take 2.5 ml and inoculate it in 250 ml medium. After culturing at 37°C overnight, transfer it to a 5L tank for fermentation. Fermentation was carried out by feeding and deep aeration culture. Glycerol or glucose was selected as the limiting carbon source before induction to control feeding, and IPTG / lactose was selected as the inducer and the limiting carbon source after induction to control feeding. The fermentation temperature was 37°C, and the pH was controlled at 6.0-7.2 by adding 25% ammonia water; the fermentation tank ventilation volume was selected to be 0.5-2.0 vvm, and the dissolved oxygen value was around 30% by controlling the feeding rate, stirring rate and ventilation volume. During the fermentation process, samples were taken regularly to measure the OD 600 , when OD 600 After reaching a certain value, IPTG / lactose was added for induction, and the fermentation was terminated after 8 to 16 hours of induction culture.

[0106] III. Drug Combinations

[0107] According to a third aspect of the present disclosure, a composition is provided, which comprises the immunoglobulin degrading enzyme as described above or a protein comprising the immunoglobulin degrading enzyme as described above, and optionally a pharmaceutically acceptable carrier or excipient.

[0108] 3.1 Antibody Targets

[0109] Preferably, in the composition as described above, the target of the antibody may be cell surface proteins, cytokines, hormones, enzymes, intracellular and intercellular messengers, immune checkpoints, etc.

[0110] 3.2 Targeted drugs

[0111] Preferably, the composition as described above, wherein the composition also includes a targeted drug or a chemotherapeutic drug or an immune checkpoint blocker.

[0112] 3.3 Drugs that can reduce blood IgG levels

[0113] Preferably, in the composition as described above, the polypeptide drug capable of reducing blood IgG levels can block the binding between blood IgG and FcRn protein.

[0114] 3.4 Viral vector drugs

[0115] Preferably, in the composition as described above, in the viral vector drug, the virus used in the viral vector drug is selected from ssDNA viruses, dsDNA viruses, ssRNA viruses or dsRNA viruses; and / or, the virus used in the viral vector drug is selected from wild-type virus strains or naturally attenuated strains, genetically engineered selectively attenuated strains, gene-loaded virus strains, and gene transcription-targeted virus strains.

[0116] 3.5 Gene therapy drugs

[0117] Preferably, in the composition as described above, the gene therapy virus expresses a foreign gene, and the foreign gene encodes a protein required for a gene defect disease.

[0118] IV. Products

[0119] The present disclosure also provides a product, which contains the immunoglobulin degrading enzyme as described above and a therapeutic agent; the therapeutic agent is selected from a viral vector drug, an antibody, and a polypeptide drug that can reduce the level of IgG in the blood.

[0120] The present disclosure also provides a kit or a kit, the kit comprising: 1) a therapeutically effective amount of a drug comprising an immunoglobulin degrading enzyme as described above; and 2) a therapeutically effective amount of a therapeutic agent; the therapeutic agent is selected from a viral vector drug, an antibody, a polypeptide drug that can reduce blood IgG levels; the viral vector drug is preferably an oncolytic virus or a gene therapy virus. The kit may also include 3) a targeted drug or a chemotherapeutic drug or an immune checkpoint blocker.

[0121] The test kit or kit comprises kit A and kit B, wherein kit A comprises a therapeutically effective amount of the immunoglobulin degrading enzyme or protein as described above, and kit B comprises a therapeutically effective amount of a therapeutic agent; the therapeutic agent is selected from a viral vector drug, an antibody, and a polypeptide drug capable of reducing blood IgG levels.

[0122] The kit may include instructions for administration of a therapeutically effective amount of an immunoglobulin degrading enzyme as described above and a therapeutically effective amount of a therapeutic agent (e.g., dosage information, dosing interval information). The therapeutic agent is selected from a viral vector drug, an antibody, and a polypeptide drug capable of reducing blood IgG levels.

[0123] The pharmaceutical carrier can be a liquid, and the pharmaceutical composition can be in the form of a solution. Liquid carriers are used to prepare solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid carrier, such as water, an organic solvent, a mixture of the two, or a pharmaceutically acceptable oil or fat.

[0124] Pharmaceutical compositions for parenteral administration are sterile, substantially isotonic, pyrogen-free, and prepared in accordance with GMP of the FDA or similar agencies. Viral vector drugs can be administered as injectable dosage forms of solutions or suspensions of the substance, wherein the substance is in a physiologically acceptable diluent and a pharmaceutical carrier (which can be a sterile liquid, such as water, oil, saline, glycerol or ethanol). In addition, auxiliary substances such as wetting agents or emulsifiers, surfactants and pH buffer substances may be present in the composition. Other components of the pharmaceutical composition are components of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil and mineral oil. Generally, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, especially for injectable solutions. Viral vector drugs can be administered in the form of accumulated injections or implant preparations, which can be formulated to allow sustained release of the active ingredient. Generally, the composition is prepared as an injectable, i.e., a liquid solution or suspension; it can also be prepared as a solid form suitable for dissolving or suspending in a liquid carrier before injection.

[0125] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred methods, devices and materials are now described.

[0126] When used in this article, the term "immunoglobulin degrading enzyme" refers to the hydrolytic cleavage of the lower hinge region of the heavy chain of the substrate immunoglobulin, effectively cutting IgG into Fc and F (ab ') 2 fragments. Any enzyme or its functional fragment. The term immunoglobulin degrading enzyme includes a variant with one or more amino acid substitutions, deletions or insertions relative to the immunoglobulin degrading enzyme SEQ ID NO: 2 sequence and / or a fusion protein or conjugate including a sialidase. Immunoglobulin degrading enzymes are also referred to as IgG degrading enzymes, and unless otherwise specified, the two terms are used interchangeably herein. When used in this article, the term "having the activity of an immunoglobulin degrading enzyme" retains, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the enzymatic activity of the polypeptide.

[0127] The term "nucleotide" or "polynucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single-strand or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically limited otherwise, the term also means oligonucleotide analogs, including PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, etc.). Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as explicitly specified sequences. In particular, degenerate codon substitutions can be achieved by generating sequences in which position 3 of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Cassol et al., (1992); Rossolini et al., Mol Cell. Probes 8:91-98 (1994)).

[0128] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, a description of a polypeptide is equally applicable to describing a peptide and describing a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the terms encompass amino acid chains of any length, including full-length proteins (i.e., antigens), in which the amino acid residues are linked via covalent peptide bonds.

[0129] The term "host cell" means a cell comprising a nucleic acid of the present disclosure, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, mating, or other methods known in the art. The exogenous polynucleotide may be maintained as a non-integrating vector such as a plasmid or may be integrated into the host genome. The host cell may be a prokaryotic cell or a eukaryotic cell.

[0130] The term "transformation" refers to a process by which a heterologous DNA sequence is introduced into a host cell or organism.

[0131] The term "expression" means the transcription and / or translation of an endogenous gene or a transgene in a cell.

[0132] The term "scale-up" broadly refers to the process of increasing a device from laboratory scale to industrial scale production. As used herein, the term includes scaling up from small shake flask expression to small fermenters or larger volume fermenters.

[0133] The term "autolysis" refers to the undesired cell lysis of a strain before the predetermined fermentation endpoint, which often results in an OD 600 The OD value of the bacterial solution decreases, the bacterial solution becomes sticky, or the turbidity of the bacterial solution increases, which has an adverse effect on the expression amount and product separation. Optionally, the "does not cause bacterial / cell autolysis" described herein is that the OD value of the bacterial solution decreases within 4 hours, 8 hours, 12 hours, 16 hours, 24 hours or longer after the bacterial expression is induced. 600 A decrease of at least 10%, at least 20%, at least 30%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% does not occur within one or more sampling points after 40, 50, 60, 70, 80, 90, 100, 110 or 120 hours. Typical OD 600 Reduce as attached Figure 3a shown.

[0134] The positive and progressive effects of the present disclosure are: it was unexpectedly found that the expression level of IdeE-1 was low and the strain was unstable, and the autolysis of the bacteria was prone to occur during the amplification culture process, and the viscosity of the fermentation liquid was extremely high, and effective solid-liquid separation could not be performed, and downstream purification operations were difficult. The present disclosure provides an immunoglobulin degrading enzyme mutant IdeE-2 and its expression method, which has the advantage of greatly improving the expression level compared to IdeE-1, and the expression system is stable, and the phenomenon of autolysis of the bacteria and viscosity of the fermentation liquid will not occur when used for amplification culture, and the final liquid volume can be arbitrarily controlled after the bacteria are broken, which is convenient for subsequent purification operations.

[0135] The present disclosure is further described below by way of examples, but the present disclosure is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are selected according to conventional methods and conditions, or according to the product instructions.

[0136] Example 1. Construction of IdeE-1 expression strain

[0137] In this embodiment, a mutant was designed based on the amino acid sequence of natural IdeE, named IdeE-1, and the amino acid sequence is shown in SEQ ID NO: 1. Through codon optimization, a polynucleotide sequence encoding the IdeE-1 protein sequence was designed and synthesized for Escherichia coli, and an N-terminal signal peptide sequence and a C-terminal 6× histidine tag were added. After the sequence was synthesized, it was inserted into the pET32a expression vector. After correct sequencing, a recombinant plasmid for expressing mutant IdeE-1 was obtained. The mutant recombinant plasmid was electrotransformed into Escherichia coli BL21 Star (DE3) and inoculated on an LB agarose plate containing 100 μg / ml ampicillin. Cultured overnight at 37°C until colonies grew. Pick a single colony, inoculate it in 3 ml of LB medium containing 100 μg / ml ampicillin, and culture it overnight at 37°C and 250 rpm. Take 500 μl of the overnight culture solution and inoculate it into 50 ml of LB medium containing 100 μg / ml ampicillin. After culturing at 37°C for 4 hours, add 0.1 mM IPTG for induction and continue induction culture overnight. The culture supernatant and cells of the overnight induced culture solution were collected by centrifugation. SDS-PAGE was used to detect the total protein in the cells and supernatant. The detection results are shown in Figure 1 As shown, lane 1 is the protein composition in the bacteria after induction culture, and lane 2 is the protein composition in the supernatant after induction culture. It can be seen that the mutant IdeE-1 is expressed in the supernatant.

[0138] Example 2. Construction of IdeE-2 expression strain

[0139] In this embodiment, another mutant was designed based on the amino acid sequence of natural IdeE, named IdeE-2, and the amino acid sequence is shown in SEQ ID NO: 2. The difference from IdeE-1 is that the N-terminus contains methionine M. Through codon optimization, a polynucleotide sequence (SEQ ID NO: 3) encoding the IdeE-2 protein sequence was designed and synthesized for Escherichia coli. After the sequence was synthesized, it was inserted into the pET32a expression vector to obtain a recombinant plasmid for expressing mutant IdeE-2. The mutant recombinant plasmid was electrotransformed into Escherichia coli BL21 Star (DE3) and inoculated on an LB agarose plate containing 100 μg / ml ampicillin. Cultured overnight at 37°C until colonies grew. Pick a single colony, inoculate it in 3 ml of LB medium containing 100 μg / ml ampicillin, and culture it overnight at 37°C and 250 rpm. 500 μl of the overnight culture was inoculated into 50 ml of LB medium containing 100 μg / ml ampicillin. After culturing at 37°C for 4 hours, 0.1 mM IPTG was added for induction and the induction culture was continued overnight. The overnight culture was collected by centrifugation, and the cells were lysed by ultrasonication. SDS-PAGE was used to analyze the total protein of the cells ( Figure 2 Lane 1 in the figure), the whole protein in the precipitate after disruption ( Figure 2 Lane 2 in the pool), and the whole protein in the supernatant after disruption ( Figure 2 Lane 3) was tested and the results were as follows Figure 2 As shown, it can be seen that the mutant IdeE-2 provided in this example is expressed intracellularly.

[0140] Example 3. Fermentation culture of IdeE-1 and IdeE-2 expression strains

[0141] The secretion and intracellular expression strains constructed in Examples 1 and 2 were gradually expanded to 5L tank fermentation culture through shake flask culture. The fermentation culture was carried out by feed addition and deep aeration culture. Glycerol or glucose was selected as the limiting carbon source before induction to control the feed addition. IPTG / lactose was selected as the inducer and the limiting carbon source after induction to control the feed addition. The fermentation temperature was 37°C, and the pH was controlled at 6.0-7.2 by adding 25% ammonia water. The fermentation tank ventilation volume was selected to be 0.5-2.0 vvm, and the dissolved oxygen value was achieved at about 30% by controlling the feed rate, stirring rate and ventilation volume. During the fermentation culture, samples were taken regularly to measure the OD 600 , when OD 600 After reaching a certain value, IPTG / lactose was added for induction, and the fermentation was terminated after 8-16 hours of induction. 600 Decreases, the bacterial solution gradually becomes sticky, and the turbidity increases significantly ( Figure 3c ), the bacterial liquid after fermentation was viscous and could not be effectively separated by centrifugation. This phenomenon is a typical autolysis phenomenon of E. coli, and multiple experiments (D1~D5) confirmed that the late autolysis phenomenon induced by this secretory expression strain was not accidental ( Figure 3a OD during the fermentation of IdeE-2 expression strain 600 Continued rise ( Figure 3b ), the protein expression in the induction cells also continued to increase with the induction time. After the cell was placed in the tank, the bacterial liquid was not sticky and the cells could be harvested by centrifugation. It can be judged that the secretory expression of IdeE-1 mutant protein in E. coli will cause the cells to autolyze, and it cannot be purified from the fermentation supernatant later, and it cannot be scaled up for production.

[0142] like Figure 4As shown, A is the protein expression result in the fermentation supernatant obtained by the IdeE-1 expression strain in a 5L tank, wherein M is a protein molecular weight standard (marker), lane 1 corresponds to the sample induced for 0 hours, lane 2 corresponds to the sample induced for 2 hours, lane 3 corresponds to the sample induced for 4 hours, lane 4 corresponds to the sample induced for 6 hours, and lane 5 corresponds to the sample induced for 8 hours. B is the protein expression result after the fermentation bacteria obtained by the IdeE-2 expression strain in a 5L tank are broken, wherein M is a protein molecular weight standard (marker), lane 1 corresponds to the sample induced for 0 hours, lane 2 corresponds to the sample induced for 2 hours, lane 3 corresponds to the sample induced for 4 hours, lane 4 corresponds to the sample induced for 6 hours, and lane 5 corresponds to the sample induced for 8 hours. It can be seen that the expression level of IdeE-2 is significantly higher than that of IdeE-1, and during the production process, the strain encoding the protein IdeE-1 by SEQ ID NO: 1 is more unstable, and the bacteria show autolysis phenomenon ( Figure 3a ), and the viscosity of the fermentation broth expressed by IdeE-1 increased, making it impossible to effectively separate the solid and liquid. The ultrafiltration liquid exchange volume was larger and more difficult, so purification was more difficult.

[0143]

[0144] Example 4. In vivo efficacy

[0145] This example evaluates the safety and tolerability, pharmacokinetic characteristics, pharmacodynamic characteristics, and immunogenicity of IdeE-2 in healthy subjects in China and New Zealand. A total of 68 healthy subjects were included in the study, and all subjects completed the study. The results of the Phase I clinical trials conducted in China and New Zealand were highly consistent, and the results showed that IdeE-2 had good safety and tolerability in healthy subjects. The PK curves of IdeE-2 in each dose group had good reproducibility, and showed the characteristics of rapid distribution and slow elimination in healthy subjects. The PK characteristics were consistent with the two-compartment model, and the optimal dose effect could be achieved by administration of 0.25 mg / kg dose. IdeE-2 can efficiently, quickly and specifically cleave human IgG. 0.25 mg / kg dose of IdeE-2 can cleave 95% of IgG within 45 minutes to 6 hours after administration, and maintain a low IgG level (average decrease of more than 70%) for 1 week ( Figure 5 ).

[0146] The applicant declares that the present disclosure illustrates the detailed method of the present disclosure through the above-mentioned embodiments, but the present disclosure is not limited to the above-mentioned detailed method, that is, it does not mean that the present disclosure must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement to the present disclosure, equivalent replacement of various raw materials of the product of the present disclosure, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present disclosure.

[0147] Sequence

[0148] SEQ ID NO: 1

[0149] ITSVWTKGVTPLTPEQFRYNNEDVIHAPYLAHQGWYDITKAFDGKDNLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPDKQKIIFNNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGDQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLS

[0150] SEQ ID NO: 2

[0151] MITSVWTKGVTPLTPEQFRYNNEDVIHAPYLAHQGWYDITKAFDGKDNLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPDKQKIIFNNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGDQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLS

[0152] SEQ ID NO: 3

[0153] ATGATCACCAGCGTGTGGACCAAAGGTGTTACCCCGCTGACCCCGGAGCAGTTCCGTTACAACAACGAAGACGTTATTCACGCGCCGTACCTGGCGCACCAAGGTTGGTATGATATCACCAAGGCGTTCGACGGCAAAGATAACCTGCTGTGCGGTGCGGCGACCGCGGGTAACATGCTGCACTGGTGGTTTGACCAGAACAAGACCGAGATTGAAGCGTACCTGAGCAAACACCCGGATAAGCAGAAGATCATCTTCAACAACCAAGAACTGTTCGACCTGAAGGCGGCGATCGACACCAAAGATAGCCAGACCAACAGCCAACTGTTCAACTATTTTCGTGATAAGGCGTTTCCGAACCTGAGCGCGCGTCAGCTGGGTGTGATGCCGGACCTGGTTCTGGATATGTTCATTAACGGCTACTATCTGAACGTGTTTAAAACCCAGAGCACCGATGTTAACCGTCCGTACCAAGACAAGGATAAACGTGGTGGCATCTTCGACGCGGTGTTTACCCGTGGTGATCAGACCACCCTGCTGACCGCGCGTCACGACCTGAAGAACAAAGGTCTGAACGATATTAGCACCATCATTAAGCAAGAACTGACCGAAGGTCGTGCGCTGGCGCTGAGCCACACCTATGCGAACGTGAGCATCAGCCACGTTATTAACCTGTGGGGTGCGGACTTCAACGCGGAGGGCAACCTGGAAGCGATCTACGTGACCGACAGCGATGCGAACGCGAGCATTGGCATGAAGAAATATTTTGTGGGTATCAACGCGCACGGCCACGTTGCGATCAGCGCGAAGAAAATTGAGGGTGAAAACATCGGCGCGCAGGTTCTGGGTCTGTTCACCCTGAGCAGCGGCAAGGATATTTGGCAAAAACTGAGCTAATGA。

Claims

1. A method for preventing an immunoglobulin degrading enzyme from causing autolysis of host cells when the immunoglobulin degrading enzyme is induced to express in host cells, characterized in that: The amino acid sequence of the immunoglobulin degrading enzyme is the amino acid sequence shown in SEQ ID NO: 2, and the host cell is an Escherichia coli cell.

2. The method according to claim 1, characterized in that During the expression of the immunoglobulin degrading enzyme, the OD of the host cell 600 Greater than 80.

3. The method according to claim 1, characterized in that During the expression of the immunoglobulin degrading enzyme, the OD of the host cell 600 Greater than 100.

4. The method according to claim 1, characterized in that: During the expression of the immunoglobulin degrading enzyme, the OD of the host cell 600 It will not decrease within 20 hours of expression.

5. The method according to claim 1, characterized in that: The immunoglobulin degrading enzyme is expressed intracellularly in host cells.

6. The method according to claim 1, characterized in that The method comprises (a) or (b): (a) transforming a nucleic acid molecule encoding an immunoglobulin degrading enzyme or an expression vector containing a nucleotide sequence encoding an immunoglobulin degrading enzyme into a host cell, culturing the host cell, and then isolating and obtaining the immunoglobulin degrading enzyme; (b) culturing a host cell containing a nucleic acid molecule encoding an immunoglobulin degrading enzyme, and then isolating the immunoglobulin degrading enzyme; or culturing a host cell containing an expression vector, and then isolating the immunoglobulin degrading enzyme, wherein the expression vector contains a nucleotide sequence encoding an immunoglobulin degrading enzyme.

7. The method according to claim 1, characterized in that The method comprises the following steps: (a) selecting a single clone of a host cell; (b) carrying out seed liquid culture on the single clone host cell; (c) transferring the single clone host cell cultured in the seed liquid to a fermenter for culture; (d) inducing the single clone host cell to express an immunoglobulin degrading enzyme; and (e) collecting the expressed immunoglobulin degrading enzyme.

8. A host cell, characterized in that The host cell comprises a nucleic acid molecule encoding an immunoglobulin degrading enzyme, or the host cell comprises an expression vector comprising a nucleotide sequence encoding an immunoglobulin degrading enzyme; The amino acid sequence of the immunoglobulin degrading enzyme is shown in SEQ ID NO: 2; The host cell is an Escherichia coli cell.

9. The host cell according to claim 8, characterized in that The immunoglobulin degrading enzyme does not cause autolysis of the E. coli cells when induced to be expressed in the E. coli cells.

10. The host cell according to claim 8, characterized in that During the expression of the immunoglobulin degrading enzyme, the OD of the E. coli cells 600 Greater than 80.

11. The host cell according to claim 8, characterized in that During the expression of the immunoglobulin degrading enzyme, the OD of the E. coli cells 600 Greater than 100.

12. The host cell according to claim 8, characterized in that During the expression of the immunoglobulin degrading enzyme, the OD of the E. coli cells 600 It will not decrease within 20 hours of expression.

13. The host cell according to claim 8, characterized in that The immunoglobulin degrading enzyme is expressed intracellularly in E. coli cells.

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